Low-molecular heparin modification and remedy for skin ulcer

ABSTRACT

The invention is direct to modified heparins, especially low-molecular weight modified heparins that are adapted for used in preparing an agent for treating a skin ulcer. The low-molecular weight modified heparins have been substantially deprived of the anticoagulant activity, yet they retain the ability to bind to cell growth factors, cytokines and cell adhesion molecules.

This is a 371 of PCT/JP98/05191 filed Nov. 18, 1998.

FIELD OF THE INVENTION

This invention relates to modified heparins and an agent for remedyingskin ulcer. More particularly, this invention relates to using as anagent for remedying skin ulcer modified heparins, especially lowmolecular weight modified heparins which have been substantiallydeprived of the anticoagulant activity yet retain the ability to bind tocell growth factors, cytokines and cell adhesion molecules, and alsorelates to modified heparins, especially low molecular weight modifiedheparins adapted for use in preparing an agent for remedying skin ulcer.

BACKGROUND OF THE INVENTION

Heparin is one of glycosaminoglycans and is characterized by havinganticoagulant activity. A lot of heparin has been found in liver, lung,intestine, spleen and other organs of healthy edible animals, andheparin is largely produced by mast cells around capillary vessels. Aheparin is glycosaminoglycan including various amounts of O-sulfate,N-sulfate and N-acetyl groups and belongs to a heteropolysaccharidehaving a molecular weight of 6,000˜20,000.

More particularly, heparin is formed by a combination of ten kinds ofdisaccharide having chemical formulas shown in the following Table 1.

In the Table, GlcA denotes D-glucuronic acid, GlcNAcN-acetyl-D-glucosamine, GlcNS N-sulfo-D-glucosamine, GIcA (20S)2-sulfo-D-glucuronic acid, GlcNS(60S) N-sulfo-D-glucsamine-6-sulfate,GlcNS(3,6diOS) N-sulfo-D-glucosamine-3,6-disulfates, IdA L-iduronicacid, IdA(20S) 2-sulfo-L-iduronic acid, respectively.

Heparin exhibits a variety of biological activities. Namely, heparinbinds to a wide variety of cell growth factors, cytokines and celladhesion molecules. The major function of heparin is that it binds toenzymes and factors such as antithrombin HI involved in bloodcoagulation and fibrinolysis, thus inhibiting blood coagulation asmentioned above.

It has never been anticipated that heparin itself is effective inremedying skin ulcer. It is because, when heparin is applied on the skinulcer, it would promote hemorrhage due to its inherent anticoagulantproperty, and accordingly it would be liable to worsen conditions of theskin ulcer.

Some trials have been made to make use of heparin as a drug bydepolymerizing heparin to form low molecular weight heparin or bychemically modifying heparin. For example, JP30277/1979 discloses thatheparin can be used as an agent for remedying thrombosis when heparin isdepolymerized to form low molecular weight heparin having molecularweights of 2,000˜5,000, and the resultant product is then chemicallymodified.

WO80/01383 discloses that a low molecular weight heparin having aselective anticoagulant activity can be obtained which has a weakenedanti-thrombotic activity and a strengthened anti-factor Xa activity,when heparin is treated with nitrite, or oxidized with periodate, andthe resultant product is subjected to β-elimination reaction withalkali.

JP66192/1988 discloses that heparin-typed oligosaccharides having anaffinity to cell growth factors and showing specific nuclear magneticresonance spectra can be used for remedying muscle and blood vesseldiseases.

WO88/06840 discloses that heparin can be used for preserving andrestoring cells when heparin is kept in an appropriate concentration.

U.S. Pat. No. 5,280,016 discloses that a heparin derivative obtained byoxidizing heparin with periodate without depolymerizing heparin and thenby reducing the oxidized heparin with borohydride can be used as a drugfor injection for inhibiting proliferation of muscle cells.

U.S. Pat. No. 5,296,471 discloses that a heparin derivative obtained byremoving 2-O-sulfate and/or 3-O-sulfate groups from heparin to variousdegrees can be used for preventing and remedying various deseases suchas cancer.

JP505179/1995 discloses that when heparin is depolymerized chemically orenzymatically to form various oligosaccharides, which are then dividedinto various fractions according to properties binding to cell growthfactors, thus divided fractions can be used for an agent for adjustinggrowth of specific cells.

WO96/29973 discloses that, when heparin is depolymerized with nitrite toform low molecular weight heparin, which is then oxidized withperiodate, and thereafter the resultant product is further reduced withborohydride, a low molecular weight modified heparin can be obtainedwhich has no anticoagulant activity, and the said modified heparin canbe used as a drug for injection for preventing thrombus formation.

WO98/14481 discloses that, when heparin is depolymerized with nitrite toform low molecular weight heparin, which is then oxidized withperiodate, and thereafter the resultant product is further reduced withborohydride, a low molecular weight modified heparin can be obtainedwhich has no anticoagulant activity, and the said modified heparin canbe used for remedying diseases such as kidney malfunction and myocardialinfarction.

As mentioned above, it is known that heparin is depolymerized to form alow molecular weight heparin, the low molecular heparin or heparinitself is chemically modified to form modified derivatives, and themodified derivatives are used for a drug. However, the drug is mainlyfor use in injection, and is used exclusively for remedying diseases ofvisceral organs such as heart and kidney.

SUMMARY OF THE INVENTION

The invention is directed to an agent for treating a skin ulcer. Theagent can be a modified heparin, especially a low-molecular weightmodified heparin. A typical agent in accordance with the presentinvention is a low-molecular weight modified heparin that has beensubstantially deprived of its anticoagulant activity, but retains itsability to bind to cell growth factors, cytokines and cell adhesionmolecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows properties of the compounds of the present invention,reference examples, and heparin.

FIG. 2 shows healing properties of the compounds of the presentinvention and reference examples.

FIG. 3 shows healing properties of the compounds of the presentinvention, heparin and Vaseline.

FIG. 4 is a microphotograph of a stained tissue specimen after it hasbeen treated with Vaseline.

FIG. 5 is a microphotograph of a stained tissue specimen after it hasbeen treated with the compounds according to the present invention.

FIG. 6 is a microphotograph of a stained tissue specimen after it hasbeen treated with the reference compound.

DISCLOSURE OF THE INVENTION

The inventors thought that, if heparin could be modified so as toeliminate or minimize the anticoagulant and antithrombotic activitiesinherent to heparin, while it maintains the ability to bind to cellgrowth factors, cytokines and cell adhesion molecules, then theresultant modified heparin would exhibit excellent effects for remedyingwounds and decubitus ulcer.

On the basis of above ideas, the inventors tried to modify heparin byoxidizing heparin with periodate, then reducing the resultant productwith borohydride to prepare a modified heparin, and thereafterdepolymerizing chemically the modified heparin so as to have an averagemolecular weight of about 1,500˜8,000, and as the result the inventorshave found that thus obtained low molecular weight modified heparin haslost the anticoagulant activity, however, retains the ability to bind tocell growth factors, cytokines and cell adhesion molecules.

Thus, the inventors mixed the low molecular weight modified heparin withappropriate base materials to prepare an ointment and applied theointment on the skin ulcer portion of a patient, and observed theprogress of the skin ulcer. As the result, the inventors have found thatthe ointment exhibits an excellent remedying effect, especially anexcellent remedying effect for decubitus ulcer. Relying on this finding,the inventors further studied and have come to complete this invention.

This invention makes it an essential feature that a specific modifiedheparin is prepared by chemically modifying heparin so as to diminish orminimize the anticoagulant activity inherent to heparin, withoutdegrading the ability to bind to cell growth factors, cytokines or celladhesion molecules inherent to heparin, and that the specific modifiedheparin is used as an agent for remedying skin ulcer. Also the inventionprovides a low molecular weight modified heparin adapted for use in theagent for remedying skin ulcer.

The term, the ability to bind to cell growth factors, cytokines and celladhesion molecules possessed by heparin, used herein means theabilities, for example, the ability to regulate activities toward immuneresponse, to exhibit ulcer healing activities, anti-virus activities,regulatory activities toward cell proliferation and differentiation, tohave interactions with proteinous factors intermediating cell-cellinteractions and binding to cytokines and cell adhesion molecules, andfurther to strengthen (or weaken in some cases) activities of thesefactors. As examples of the proteinous factors may be given interferonγ, interleukins, tumor necrotic factors, lymphotoxing, colonystimulating factors, epidermal growth factors, fibroblast growthfactors, transforming growth factor β, endothelial cell growth factors,hepatocyte growth factors, laminin and fibronectin.

The term, anticoagulant activity, used herein means the activity ofinhibiting blood coagulation. The anticoagulant activity can beindicated by antithrombotic activity, which may be measured by APTT(Activated Partial Thromboplastin Time), and also by anti-factor Xaactivity, which can be measured by specific assay for anti-factor Xaactivity. Methods for measuring these activities are fully explained,for example, by T. W. Barrowcliffe in his statements (Heparin assays andstandardization in Heparin, D. A. Lane and U. Lindahl ed. Edward Arnold,1989, P.393-415), by which the anticoagulant activity can be known.

The invention will be fully described hereunder, in which FIG. 1 showsresults of analysis of the compounds obtained in Example 1, Example 2,Example 3, Reference Example 1 and Reference Example 2 compared withheparin by High Performance Liquid Chromatography (HPLC); FIG. 2 showscomparison of days required for remedying skin ulcer in Experiment 1using the compounds obtained in Example 1, Example 2, Example 3 andReference Example 2; FIG. 3 shows comparison of days required forremedying skin ulcer in Experiment 2 using vaseline, heparin and thecompounds obtained in Reference Example 1; FIG. 4 shows amicrophotograph of a stained tissue specimen after it has been treatedwith vaseline alone for 11 days in Experiment 1; FIG. 5 shows amicrophotograph of a stained tissue specimen after it has been treatedwith the compounds obtained in Examples 1, 2 and 3 for 11 days inExperiment 1; and FIG. 6 shows a microphotograph of a stained tissuespecimen after it has been treated with the compound obtained inReference Example 2 for 11 days in Experiment 2.

In the invention, use for remedying skin ulcer may be made of a modifiedheparin obtained by modifying heparin, or a low molecular weightmodified heparin obtained by depolymerizing heparin and modifying theresultant low molecular weight heparin. The depolymerization andmodification may be carried out in either order. The low molecularweight modified heparin may be one which is prepared by chemicallymodifying low molecular weight heparin available in the market, or onewhich is prepared by at first chemically modifying heparin, and thendepolymerizing the resultant modified heparin.

In the invention, a method was adopted for preparing the low molecularweight heparin, in which heparin was at first chemically modified tolose and forfeit the blood coagulating property, and thereafter theresultant product was depolymerized to form low molecular weightcompounds. For chemical modification was used the conventional method inwhich heparin was oxidized at first with periodate, and then theresultant product was reduced with borohydride. As is well known, theperiodate attacks generally a portion including vicinal hydroxyl groups,or an aminoalcohol portion to form two aldehyde groups (For example,L.-A. Fransson et al., Carbohydr. Res.,80, 131-145, 1980). Whenperiodate is reacted with heparin, hexuronic acid residues (glucuronicacid and iduronic acid) having no sulfate group included in heparin areoxidized, as shown by chemical formulas in Table 2. Degree of oxidationis dependent on concentration of reagents, pH, temperature and time whenthe reaction is carried out.

When oxidation is carried out with periodate, pH is critical. At low pHsuch as 3, iduronic acid residues are preferentially oxidized, however,at high pH such as more than about 5, glucuronic acid residues are alsooxidized. In order to eliminate anticoagulant activity of heparin,glucuronic acid residue should be oxidized, and in the invention pH ismaintained at 3˜6, preferably 5. Thus, compound (c) shown in Table 2 canbe obtained, which has aldehyde groups. When the compound (c) is furtherreduced with borohydride, compound (a) shown in Table 2 can be obtained.The compound (a) has no blood coagulant activity (B. Basu et al.Arzneim. -Forsh./Drug Res., 36, 637-642, 1986). The compound (a) isdisclosed in U.S. Pat. No. 5,280,016.

In Table 2, oxidation and reduction are shown in respect of iduronicacid, which is taken as an example, and these reactions can be carriedout in the same manner as for glucuronic acid. The heparin modificationrepresented by chemical formula (a) is depolymerized to form lowmolecular weight compounds under acidic conditions, and the lowmolecular weight compounds having appropriate molecular weights can beobtained by controlling pH, temperature and reaction time. Both modifiedheparins represented by chemical formulas (a) and (c) have an activityfor remedying skin ulcer, however, it is found that the activity isinferior to that of low molecular weight modified heparin prepared bydepolymerizing the modified heparin.

On the other side, the compound (c) produced by periodate oxidationundergoes β-elimination reaction to form low molecular compound (b) asshown in Table 2 under the strong alkaline conditions, and the compound(b) is further decomposed. Thus obtained low molecular weight modifiedheparin (d) can be also used for an agent for remedying skin ulcer.

As mentioned above, both modified heparins shown by chemical formulas(a) and (c) can be used for an agent for remedying skin ulcer. However,these modified heparins, when compared with their depolymerized ones,are inferior in the effects for remedying ulcer as already mentionedabove.

In the invention, the modified heparin shown by the chemical formula (a)in Table 2 is further depolymerized to form low molecular weightmodified heparins (e) and (f), which retain the ability to bind to cellgrowth factors, cytokines and cell adhesion molecules, and which forfeitsubstantially anticoagulant activity.

For chemical depolymerization of heparin may be used various methodswhich include hydrolysis, deaminative degradation, oxidative degradationwith periodate, oxidation and reduction induced by radicals, degradationwith sulfuric acid and degradation by elimination reaction (K. Nagasawa,Carbohydrate Technology, Kabushiki Kaisha Sangyo Chohsakai,Biotechnology Information Center, August 1992, P.315-342).

In the invention, hydrolysis and deaminative degradation are adopted fordegradation to form low molecular weight compounds. By either of theseprocedures, satisfactory results can be obtained if the procedures areadvanced under properly controlled conditions.

The hydrolysis is carried out under acidic conditions, for example, thehydrolysis can be completed in 3 hours if pH and temperature aremaintained at 2 and 60° C., respectively. Thus, as shown in the leftlower part in Table 2, the compound (a) can be cleaved ultimately at thesite of every hexuronic acid. Therefore, when the hydrolysis is stoppedat an appropriate point by shortening the reaction time and/or loweringthe reaction temperature, it is possible to obtain low molecular weightmodified heparins (e) and (f) having desired average molecular weights.

Thus obtained compound (e) has at its end L-threonic acid, which is anaglycon of the aminosugar at the reducing terminal of the compound (e),and the carboxyl group of the L-threonic acid may be further modified byesterification, amidation and so on to form secondarily modifiedcompounds. The secondarily modified compounds are also included in thelow molecular weight modified heparins.

The deaminative degradation is carried out as shown in Table 3 usingnitrite under acidic conditions. The nitrite reacts with N-sulfate groupin aminosugar at low pH to form an unstable N-nitroso compound as anintermediate which then cyclizes intramolecularly to form2,5-anhydro-D-mannose, and the glycosidic bond of glucosamine iscleaved. Reaction of the nitrite with N-sulfate group of the glucosamineproceeds at the fastest speed at pH 1.5, at gradually decreased speedaccording as pH is elevated, and is substantially stopped when pH is 4or higher. Therefore, by controlling concentration (amount) of nitrite,reaction time, pH and reaction temperature, it is possible to obtain alow molecular weight modified heparin having a desired average molecularweight.

The low molecular weight heparin obtained by reacting heparin withnitrite has at its reducing terminal an aldehyde group of2,5-anhydro-D-mannose, which may be modified chemically in various waysas shown in below Table 4. For example, the aldehyde group may bereacted with compounds having amino group to form a Schiff base. Alsothe aldehyde group may be reduced to an alcohol group using anequivalent amount of sodium borohydride in a medium of water or alcoholin the presence of sodium bicarbonate at room temperature or under icechilled conditions. Also the aldehyde group may be oxidized to form acarboxyl group using 1-5 equivalents of active manganese dioxide in amedium of water or alcohol in the presence of Celite. Further thusobtained carboxyl group may be esterified or amidated to producecorresponding ester or amide derivatives, respectively, or the carboxylgroup may be subjected to reductive amidating reaction by usingborohydride in the presence of amine to produce corresponding variousaminomethyl derivatives. These secondarily modified heparins obtainedfrom the modified heparin have good effect for remedying skin ulcer. Thesecondarily modified heparins, therefore, are included in the lowmolecular weight modified heparin.

It is preferable that the low molecular weight modified heparins have anaverage molecular weight of 1,500˜8,000, preferably 2,000˜6,000. Themodified heparin, original heparin, and low molecular weight modifiedheparins (for example, the low molecular modified heparins obtained inExamples 1-3 mentioned below) can be clearly distinguished from oneanother by means of high performance liquid chromatography (HPLC).

FIG. 1 shows results of HPLC, wherein a column, TSK, G-2000 SWXL (0.78cm in diameter, 30 cm in length) prepared by Tosoh Corporation is used,and silicone resin fabricated beforehand to have a property of molecularsieve is filled in the column. The column has the ability to distinguisheffectively compounds having a molecular weight of 500˜20,000 when thecompounds have generally linear molecular shapes like heparin.

The column was connected with a chromatopack C-R4A of HPLC 6A seriesmade by Shimadzu Corporation. For the solvent was used 0.01M phosphatebuffer solution of pH 7.3 containing 0.15M NaCl, which was developed ata rate of 0.5 ml per minute. The development was carried out at roomtemperature, and light of 210 nm was applied to the resultant eluates todetect heparin and its derivatives by measuring absorption of the light.

FIG. 1 is a graph in which the abscissa represents time (expressed byminutes) and the ordinate represents a relative intensity of absorptionof light of 210 nm. In FIG. 1, a line including square marks showsbehavior of heparin, a line including circle marks shows behavior of themodified heparin (which was obtained in Reference Example 2), a lineincluding triangle marks shows behavior of the low molecular weightheparin (which was obtained in Reference Example 1) and a thick lineincluding no marks shows behavior of the low molecular weight modifiedheparins (which were obtained in Examples 1, 2 and 3). The low molecularweight modified heparin is eluted in the area of molecular weight ofabout 1,500˜8,000. As shown in FIG. 1, the low molecular weight modifiedheparins can be clearly distinguished from the original heparin andmodified heparins. In addition, in FIG. 1, the modified heparin obtainedin Reference Example 2 behaves as if it were a compound having somewhatlower molecular weight. However, it is assumed that this was caused bythe probable facts that molecular shape of the modified heparin wasslightly curved due to opening of ring structure in hexuronic acid. Apeak observed at an elution time of 22 minutes shows existence of thesalts.

Thus obtained modified heparin or low molecular modified heparin areseparated and refined in appropriate methods known in itself and can beused as a remedying agent. For separating and refining can be usedvarious methods such as precipitation methods using an organic solvent(alcohol, acetone etc.), various chromatography methods (for example,chromatographies using an ion exchange resin, active carbon, antibody orSepharose) and refining methods by means of molecular sieves.

In the invention, both the modified heparin which was not depolymerizedand was obtained in Reference Example 2, and the modified heparin whichwas depolymerized to have low molecular weights can be used as the agentfor remedying skin ulcer. However, it is preferable to use thedepolymerized modified heparin.

In case wherein heparin is depolymerized by a hydrolysis method, aproduct is obtained having L-threonic acid as an aglycon of aminosugarat the reducing terminal thereof, and aminosugar at the non-reducingterminal thereof, and in case wherein heparin is depolymerized bydeaminative degradation, a product is obtained having2,5-anhydro-D-mannose at the reducing terminal thereof and uronic acidat the non-reducing terminal thereof.

In the invention, the modified heparin and low molecular weight modifiedheparin (hereinafter both are collectively referred to as a heparinderivative) can be used as they are or after they were changed intosalts thereof. The salts may have whichever forms, if the salts may beadmitted pharmacologically. For example, the salts may be formed withorganic acids such as acetic acid or inorganic acids such ashydrochloric acid. The salts may be formed by changing an acid groupsuch as a sulfate residue in the heparin derivative into an alkali oralkaline earth metal salt. Also the heparin derivative may be a singlecompound or a mixture thereof.

The heparin derivative in the invention is effective for remedyingvarious skin ulcers, for example, destruction, deficit etc. of skincaused by necrosis, desquamation, melting etc. of skin, moreparticularly, skin ulcers caused by wounds, decubitus, scalding,frostbite, or operation wounds, and skin infections (e.g. skin mycosis,psoriasis, varicella, tinea pedis, tinea corporis, pimple etc.).

The heparin derivative according to the invention can be administeredparenterally or perorally as the agent for remedying skin ulcers.Preferably, it is administered parenterally. In case of parenteraladministration, it may be used in the form of percutaneousadministration drugs, that is, it may have any forms if it can beapplied directly to affected parts of skin. It may be in the form ofdrugs able to provide active ingredients percutaneously, preferably,patch, cataplasm, ointment (including cream), plaster, tape, lotion,liquid, suspension, emulsion, aerosol (including sprayed materials) andso on. Applying articles such as patch, cataplast, tape, plaster,ointment and sprayed material are most preferable in respect that theactive ingredients can be readily controlled.

The ointment, lotion, liquid, suspension, emulsion and aerosol can beprepared by blending the active ingredients with solvent, suspendingagent, emulsifier, aerosol and base, respectively, which are known perse. At this time, antiseptics (for example, ethyl ρ-hydroxybenzoate,benzalkonium chloride etc.) may be further added, if desired.

Also the patch, cataplast, plaster and tape can be prepared by using abase known in itself in the pharmaceutical field, forming a mixture ofthe base and the active ingredients, adding thereto antiseptics ifdesired, and making the mixture absorbed in or adhered to an appropriatecarrier. For the carrier can be used a high polymer membrane (forexample, polyethylene, ethylene-vinyl acetate copolymer, polyethyleneterephthalate etc.), woven fabric, non-woven fabric, paper, aluminiumfoil etc. For adhesives for forming the patch, cataplast and tape can beused various adhesives belonging to polyalkylvinylether,polyalkylacrylate, polyisobutylene, natural rubber and synthetic rubber.Further, animal oil (for example, squalene or squarane), vegetable oil(for example, olive oil or jojoba oil), vaseline, lanolin etc. may beadded in order to give appropriate plasticity and tackiness.

When percutaneous drugs are prepared, such as ointment, plaster, tape,patch, cataplast and so on, an ingredient for regulating percutaneousabsorption can be added. This ingredient can include lipids andfat-soluble substances including phospholipid such as lecithin, solidparaffin, beeswax, carnauba wax, hardened castor oil, lanolin, Vaseline®(petroleum jelly), polyvinyl alcohol, polyvinyl pyrrolidone,polyethylene glycol, fatty acid glycerol ester, cholestrol, fatty acidshaving about 6-22 carbons, (for example, capric acid, caprylic acid,caproic acid, lauric acid, myristic acid, palmitic acid, stearic acid,arachidonic acid, etc.) and their salts, aliphatic alcohol having about6-22 carbons (for example, n-octyl alcohol, n-cetyl alcohol, stearylalcohol etc.), silicone resin, and low aliphatic alcohol (for example,ethanol, isopropyl alcohol etc.). Of course, the active ingredients maybe used alone or together with more than two of these bases.

The solvent can be water, low aliphatic alcohols (for example, ethanol,etc.), alkane diol having about 2-6 carbons (for example, glycol, etc.),and alkane triol having about 3-7 carbons (for example, glycerol). Thesuspending agent and emulsifier can be gum arabic, carboxymethylcellulose, methyl cellulose, sodium alginate, etc. The base of theointment, tape, patch and cataplasm can be Vaselineg (petroleum jelly),solid paraffin, vegetable oil, mineral oil, lanolin, wax, macrogol, etc.The base of plaster can be beeswax, paraffin, macrogol, glycerol esterof fatty acid, etc.

For a spraying agent can be used incombustible liquefied gas (forexample, Freon 11, Freon 12, Freon 13 etc.).

In case of oral administration, the heparin derivative may beadministered in the form of tablet, capsule, powder, granule, syrup,emulsion and suspension, which can be made according to the conventionalmethods.

An amount of the heparin derivative contained in the agent for remedyingskin ulcer according to the invention is not limited and can be variedwithin the range wherein the agent can exhibit desirable remedyingeffects. The amount is dependent on the object, whichever it may behuman or animal, to which the agent is administered, and also on thekind of diseases and the degree of diseases. For example, the amount ofheparin derivative, acting as the active ingredient, contained in theagent for remedying decubitus according to the invention isapproximately 0.001˜30%, preferably approximately 0.01˜10%, morepreferably approximately 0.05˜5% by weight of the total weight of thepercutaneous drug, and in case of the patch, plaster, tape etc., anamount of heparin derivative per unit area is approximately 0.1 mg˜200mg/cm², preferably approximately 1˜mg 20 mg/cm². Administrationfrequency is varied dependent on the kind and conditions of diseases.For example, the percutaneous application or administration is made fromonce to several times per day, and the application or administration iscontinued for more than two days.

The agent for remedying skin ulcer according to the invention maycomprise other pharmaceuticals as an effective ingredient so long as theagent and other pharmaceuticals do not deteriorate their mutualremedying effects. The other pharmaceuticals may be any one, if it doesnot hinder the effects of the agent for remedying skin ulcer, andinclude, for example, various cell growth factors, cytokines or celladhesion molecules (preferably transforming growth factor β,vasoendothelial cell growth factors, fibroblast growth factors orhepatocyte growth factors), various antibiotics (antibiotic agents,antifungal agents, antiviral agents etc.) various steroids,anti-inflammatory agents, anti-allergic agents, anti-histamic agentsetc.

For lipids can be used various compounds, for example, phospholipids,preferably lecithin, and for fat-soluble substances can be used, forexample, animal oils (e.g. squalene, squarane etc.), vegetable oils(e.g. olive oil, jojoba oil etc.), solid paraffin, beeswax, carnaubawax, hardened castor oil, lanolin, vaseline, polyvinyl alcohol,polyvinyl pyrrolidone, polyethyleneglycol, fatty acid glycerol ester,cholesterol, aliphatic carboxylic acid having about 6˜22 carbons (e.g.capric acid, caprylic acid, caproic acid, lauric acid, myristic acid,palmitic acid, stearic acid, arachidonic acid etc.) and their salts,aliphatic alcohol having about 6˜22 carbons (e.g. n-octyl alcohol,n-cetyl alcohol, stearyl alcohol etc.), preferably vaseline,polyethylene glycol, and aliphatic alcohol having about 6˜22 carbons(e.g. n-octyl alcohol, n-cetyl alcohol, stearyl alcohol etc.), morepreferably vaseline, polyethylene glycol and stearyl alcohol.

The agent for remedying skin ulcer according to the invention is verylow in toxicity, and in case wherein the agent has been administered fora long period, either side effects or toxicity are hardly recognized.Therefore, the percutaneous drugs containing the heparin derivativeaccording to the invention can be administered in safety for remedyingthe skin ulcer mentioned above, especially for remedying decubitus.

The drugs characterized by comprising the heparin derivative accordingto the invention can be simply and easily administered to patients, andwhen used for remedying decubitus, the drugs can reduce greatly theburden of patients and nurses not only in hospitals but also at home.

The invention is fully explained below by way of Reference Examples,Examples and Experiments. However, these are offered only forillustrative purposes, and do not limit the invention.

MANNERS OF EMBODIMENTS Reference Example 1

One gram of porcine heparin was dissolved in 12.5 ml of distilled water,and thereto was added 1.25 ml of 5% sodium nitrite aqueous solution atroom temperature. Then thereto was added 1.25 ml of 33% acetic acidaqueous solution, and the resultant solution was stirred for 50 minutesat room temperature to form a reaction solution. To the reactionsolution were added 4 ml of 1M sodium carbonate aqueous solution and 1ml of 1M sodium hydroxide aqueous solution in this order, and pH of thereaction solution was adjusted to 9.0. Thereto was further added 0.3 mlof 0.01M sodium hydroxide aqueous solution containing 0.25M sodiumborohydride, and the resultant solution was allowed to stand for 30minutes at 50° C. Then by adding glacial acetic acid thereto excessivesodium borohydride was decomposed, and subsequently the solution waspassed through a column of Sephadex G-25 which had been equilibratedwith 0.3M sodium acetate solution. The resultant high molecular weightfractions were concentrated under reduced pressure, and then by addingethanol thereto an aimed product was precipitated. The precipitate wascollected by filtration and dried to obtain about 0.9 g of low molecularweight heparin which had some anticoagulant activity.

Bovine heparin was subjected to similar operations and a low molecularweight heparin could be obtained which had some anticoagulant activity.Thus obtained low molecular weight heparin was subjected to HPLCanalysis, in which it showed behavior as indicated by the line includingtriangle marks in FIG. 1.

Reference Example 2

One gram of porcine heparin was dissolved in 20 ml of 0.05M acetatebuffer solution (pH 5.0) containing 0.1M sodium periodate, and theresultant solution was allowed to stand in the dark for 72 hours at 4°C. By adding glycerol thereto excess periodate was decomposed, and thenthe resultant solution was dialyzed against distilled water for threedays at about 5° C., using a semipermeable membrane for use in dialysis(manufactured by Spectrum Co., Code Number 530-3518, MW500cut). Thedialyzed solution was subjected to lyophilization, and the resultantproduct was dissolved in 0.25M sodium bicarbonate aqueous solution (pH9.5) containing 0.2M sodium borohydride to make a 10% solution. Thesolution was allowed to stand for 3 hours at 4° C. By adding glacialacetic acid to the solution excess sodium borohydride was decomposed,and the solution was adjusted to have pH 5, and allowed to stand for 30minutes. Subsequently the solution was neutralized with 0.1M sodiumhydroxide aqueous solution. The resultant solution was again dialyzedagainst distilled water at about 5° C. for 3 days, using thesemipermeable membrane for use in dialysis, and then the resultingsolution was subjected to lyophilization to obtain 0.85 g of lyophilizedproduct of modified heparin.

Bovine heparin was treated in similar manners and a modified heparinaccording to the invention could be obtained.

Thus obtained modified porcine heparin was subjected to HPLC analysis,in which it showed behavior as indicated by the line including circlemarks in FIG. 1, in which the modified heparin behaved as if it were ofslightly lower molecular weight, when compared with the originalheparin. However, it is considered that this was probably caused bydeformation in molecule of the modified heparin, because any changes inmolecular weight cannot occur in the modification process mentionedabove.

Example 1

One gram of the low molecular weight heparin obtained from porcineheparin by the method described in Reference Example 1 was dissolved in0.05M acetic acid buffer solution (pH 5.0) containing 20 ml of 0.1Msodium periodate, and the resultant solution was allowed to stand in thedark for 72 hours at 4° C. By adding glycerol to the solution excessperiodate was decomposed and then the solution was dialyzed againstdistilled water for 3 days at about 5° C., using a semipermeablemembrane for use in dialysis (manufactured by Spectrum Co., Code Number530-3518, MW500cut). The dialyzed solution was subjected tolyophilization, and the resultant product was dissolved in 0.25M sodiumbicarbonate aqueous solution (pH 9.5) containing 0.2M sodium borohydrideto make a 10% solution. The solution was allowed to stand for 3 hours at4° C. By adding glacial acetic acid thereto excessive sodium borohydridewas decomposed, thereafter the solution was adjusted to have pH 5.0 andallowed to stand for 30 minutes. Subsequently the solution wasneutralized with 0.1M sodium hydroxide aqueous solution. The resultantsolution was again dialyzed against distilled water at about 5° C. for 3days, using the semipermeable membrane for use in dialysis, and then theresultant solution was subjected to lyophilization to obtain 0.7 g oflyophilized product, which was a low molecular weight modified heparinaccording to the invention which has no anticoagulant activity.

Bovine heparin was treated in similar manners, and a low molecularweight modified heparin according to the invention could be obtained.

Thus obtained low molecular weight modified porcine heparin wassubjected to HPLC analysis, in which it showed such behavior asindicated by a thick line including no mark in FIG. 1, in which it isclear that the low molecular weight modified heparin had a lowermolecular weight when compared with the original heparin and themodified heparin obtained in Reference Example 2. The molecular weightis seemingly in the range from about 1,500˜8,000.

Example 2

One gram of porcine modified heparin obtained by the method described inReference Example 2 was dissolved in 20 ml of 0.1M hydrochloric acidaqueous solution, and the resultant solution was carefully adjusted tohave pH 2. The solution was heated rapidly to 60° C., and allowed tostand for about 1 hour. Immediately thereafter while being ice-chilled,the solution was neutralized with 0.1M sodium hydroxide aqueoussolution, and then dialyzed against distilled water for 3 days at about5° C., using a semipermeable membrane for use in dialysis (manufacturedby Spectrum Co., Code Number 530-3518, MW500Cut). The dialyzed solutionwas lyophilized to obtain 0.7 g of a low molecular weight modifiedheparin according to the invention.

Bovine modified heparin was treated in similar manners, and a lowmolecular weight modified heparin according to the invention could beobtained.

Thus obtained low molecular weight modified porcine heparin wassubjected to HPLC analysis, in which it showed such behavior asindicated by a thick line including no mark in FIG. 1, and the behaviorwas the same as that of the low molecular weight modified heparinobtained in Example 1.

Example 3

One gram of porcine modified heparin obtained by the method described inReference Example 2 was dissolved in 12.5 ml of distilled water, andthereto was added 1.25 ml of 5% sodium nitrite aqueous solution. Thenwhile being stirred, thereto was added 1.25 ml of 33% acetic acidaqueous solution and the resultant solution was further stirred for 50minutes at room temperature. Thereto were added 4 ml of 1M sodiumcarbonate aqueous solution and 1 ml of 1M sodium hydroxide aqueoussolution in this order, and the resultant solution was adjusted to havepH 9.0. Thereto was added 0.3 ml of 0.01M sodium hydroxide aqueoussolution containing 0.25M sodium borohydride, and the resultant solutionwas allowed to stand for 30 minutes at 5° C. Subsequently, by addingglacial acetic acid thereto excessive sodium borohydride was decomposed,and then the resultant solution was either passed through a columnfilled with Sephadex G-25 equilibrated with 0.3M sodium acetate aqueoussolution, or dialyzed in the same manners as in Examples 1 and 2. Theliquid which had passed through the column (fractions containing highermolecular weight compounds), or the dialyzed solution was concentrated,and thereafter by adding ethanol thereto a low molecular weight modifiedheparin was precipitated. The precipitate was dried to obtain 0.9 g of adried product.

Bovine modified heparin was treated in the same manners, and a lowmolecular weight modified heparin could be obtained.

The low molecular weight modified porcine heparin obtained herein wassubjected to HPLC analysis, in which it showed such behavior asindicated by a thick line including no mark, and the behavior wasseemingly the same as that of the low molecular weight modified heparinsobtained in Examples 1 and 2.

Example 4

Below mentioned compounds were mixed as described below.

The low molecular weight modified 1 part heparin obtained in any of byweight Examples 1 ˜ 3 Stearyl alcohol 33 parts by weight Propyleneglycol 33 parts by weight Polyalkylvinyl ether 33 parts by weight

The low molecular weight modified heparin was mixed with propyleneglycol, then stearyl alcohol was added thereto, and the resultantmixture was blended and kneaded to form an ointment material, to whichwas added and blended polyalkylvinylether acting as a tackifier to forman ointment.

The ointment was applied on a release liner made of a polyester resin toform an ointment layer of about 0.1 mm in thickness, thereafter theointment layer was transferred to a polyester film to obtain a plastercontaining 1%(W/W) low molecular weight modified heparin.

The plaster was cut to a size fit for an affected part, and can be usedfor therapy.

Example 5

Below mentioned compounds were mixed as described below.

The low molecular weight modified 1 part heparin obtained in any of byweight Examples 1 ˜ 3 Vaseline 69 parts by weight Polyalkylvinyl ether30 parts by weight

The compounds were mixed intimately to form a homogeneous ointment,which was then applied on a flexible gauze (woven fabric) to obtain aplaster having an agent layer of about 0.1 mm in thickness andcontaining 1%(W/W) low molecular weight modified heparin.

The plaster was cut to a size fit for an affected part, and can be usedfor therapy.

Example 6

The low molecular weight modified 1 part heparin obtained in any of byweight Examples 1 ˜ 3 Vaseline 99 parts by weight

These compounds were mixed intimately to obtain a homogeneous ointment.The ointment was applied to an affected part just to cover the part inan appropriate thickness, and can be used for therapy.

Experiment 1

Mouse (including diabetic mouse), rat, quinea pig and mini-pig were usedto test the materials described in the present invention for remedyingeffect for skin ulcer. This experiment is in the case wherein thematerial was applied to the quinea pig.

Skin ulcers having a diameter of 8 mm and a depth of 2˜3 mm were formedat three spots on each side of back bone of quinea pigs weighing about200˜300 g using a metal punch. An ointment was prepared by adding thelow molecular weight modified heparin obtained in any of Examples 1˜3 tovaseline in a ratio of the heparin 1 mg/the vaseline 1 g and mixingintimately the resulting mixture, and 1 g of the ointment was applied tothree skin ulcers on one side, and for the purpose of comparison 1 g ofvaseline containing no drug was applied to three skin ulcers on theother side. The whole back of each of the quinea pigs was covered with atransparent dressing film (10 cm×12 cm), which was fixed by a flexibletape.

Effectiveness of the agent was assayed by two methods, one of which is(i) comparison of the number of days up to complete closure of theulcers and the other is (ii) analyzing tissue specimen of the ulcersafter it had been stained (with hematoxylin-eosin).

As the results, it was found that (i) as seen from FIG. 2, 22±2 dayswere required for apparent healing (skin surface closure) when vaselinealone was used. However, significantly shortened periods such as only16±2 days were required for the apparent healing, when any one of thelow molecular weight modified heparins obtained in Examples 1˜3 wereused. When the modified heparin in Reference Example 2 was used, 17±2days were required for apparent healing, and these results were at aglance equal to those in case wherein the low molecular weight modifiedheparins obtained in any of Examples 1˜3 were used. However, when thetissues were dissected and inspected, the equality was found to benothing but apparent phenomena. (ii) when tissue specimens were taken onthe 11th day and stained, the following differences were found: Whenvaseline alone was used, the specimen did not show restoration of skinsurface and skin epithelization did not occur as shown in FIG. 4. Incontrast, when the low molecular weight modified heparins obtained inany of Examples 1˜3 were used, both corneum and appendage structureswere formed and almost complete skin epithelization was recognized asshown in FIG. 5. On the other side, when the modified heparin obtainedin Reference Example 2 was used, the healing effect was considerablyinferior to that when the low molecular weight modified heparins wereused, because as shown in FIG. 6, both corneum and appendage structureswere insufficiently formed, although the skin surfaces were seeminglyrestored. From these results it was confirmed that the low molecularweight modified heparins according to the invention were especiallyeffective in remedying skin ulcers.

Experiment 2

In the same manners as in Experiment 1, heparin and low molecular weightmodified heparin obtained in Reference Example 1 having anticoagulantactivity were tested for remedying skin ulcers. As the results nosignificant effects were found in any of two experiments, as far as daysup to complete remedy (FIG. 3) and skin epithelization in tissuespecimens are concerned.

INDUSTRIAL APPLICABILITY

The modified heparins and low molecular weight modified heparinsaccording to the present invention have excellent effects for remedyingskin ulcers such as skin wounds, especially decubitus ulcers, and alsohave such low toxicity that they give neither side effect nor toxicityeven if administered for a long term, so that they are useful as anagent for remedying skin ulcers.

What is claimed is:
 1. A composition for treating skin ulcer comprisingan effective amount of a modified heparin for remedying skin ulcer in apharmaceutically acceptable carrier selected from the group consistingof petroleum jelly and polyalkylvinylether for enhancing cutaneousabsorption, wherein the modified heparin is prepared by oxidizingheparin with periodate and then reducing the resultant product withborohydride; and the modified heparin is substantially deprived ofanticoagulant activity but retains an ability to bind to cell growthfactors, cytokines and cell adhesion molecules.
 2. A composition fortreating skin ulcer comprising an effective amount of a modified heparinfor remedying skin ulcer in a pharmaceutically acceptable carrierselected from the group consisting of petroleum jelly andpolyalkylvinylether for enhancing cutaneous absorption, wherein themodified heparin has an average molecular weight of about 1,500 to8,000; the modified heparin is prepared by oxidizing heparin withperiodate, and then reducing the resultant product with borohydride, andsubsequently depolymerizing the reduced heparin; and the modifiedheparin is substantially deprived of anticoagulant activity but retainsan ability to bind to cell growth factors, cytokines and cell adhesionmolecules.
 3. A composition for treating skin ulcer comprising aneffective amount of a modified heparin for remedying skin ulcer in apharmaceutically acceptable carrier selected from the group consistingof petroleum jelly and polyalkylvinylether for enhancing cutaneousabsorption, wherein the modified heparin is prepared by depolymerizingheparin, oxidizing the depolymerized heparin with periodate, and thenreducing the oxidized heparin with borohydride; and is substantiallydeprived of anticoagulant activity but retains an ability to bind tocell growth factors, cytokines and cell adhesion molecules.
 4. Thecomposition for treating skin ulcer according to claim 3, wherein thecomposition is a percutaneous composition and the modified heparin hasan average molecular weight of about 1,500 to 8,000.
 5. A compositionfor treating skin ulcer comprising an effective amount of a modifiedheparin for remedying skin ulcer in a pharmaceutically acceptablecarrier, wherein the modified heparin: has an average molecular weightof about 1,500 to 8,000; is prepared by oxidizing heparin withperiodate, and then reducing the resultant product with borohydride,subsequently depolymerizing the reduced heparin, and further modifyingactive groups located at terminals of the depolymerized heparin to amember selected from the group consisting of carboxyl group, ester ofcarboxyl group, amide of carboxyl group, and Schiff bases; and issubstantially deprived of anticoagulant activity but retains an abilityto bind to cell growth factors, cytokines and cell adhesion molecules.6. The composition for treating skin ulcer according to claim 1, whereinthe modified heparin is present in an amount of 0.001 to 30% by weightof the composition.
 7. The composition for treating skin ulcer accordingto claim 2, wherein the modified heparin is present in an amount of0.001 to 30% by weight of the composition.
 8. The composition fortreating skin ulcer according to claim 3, wherein the modified heparinis present in an amount of 0.001 to 30% by weight of the composition. 9.The composition for treating skin ulcer according to claim 4, whereinthe modified heparin is present in an amount of 0.001 to 30% by weightof the composition.
 10. The composition for treating skin ulceraccording to claim 5, wherein the pharmaceutically acceptable carrier isa member selected from the group consisting of petroleum jelly andpolyalkylvinyleither, and the modified heparin is present in an amountof 0.001 to 30% by weight of the composition.